| In mammals, sex determination and differentiation is determined by the antagonistic and balancing actions between male and female pathway genes. Meanwhile, coordination between follicles and oocytes are indispensable for ovarian differentiation. Primitive follicles is the basic unit of reproductive system in ovaries, many transcription factors that regulate the formation of follicle and the embryonic work at the early development of follicular. Mouse Figla (factor in the germ line, alpha), encoding a germ cell-specific basic helix-loop-helix transcription factor, was first identified as an activator of oocyte genes, which plays a crucial role in the formation of primordial follicles. Figla was first detected in oocytes at E13.5 in mice, which regulates the expression of multiple oocytes pecific genes, such as the zona pellucida (Zp) proteins and the formation of the Zona pellucid, At the same time, Figla can repress sperm-associated genes during postnatal oogenesis in mammals. Tilapia (Oreochromis niloticus), an economically important species for aquaculture with XX/XY sex determination system, is known to be an ideal model to study fish sex determination/differentiation due to the availability of monosex larvae, genome information and well-documented sexual development processes. In this study, real-time PCR and immunohistochemistry were performed to study the expression of Figla in tilaipa gonads. In addition, the function and mechanisms of Figla in tilaipa sex determination and differentiation was elucidated by transgenic over-expression and knockout. The main results are listed as follows:1) The ORF (Open Reading Frame) of Figla gene which encodes a polypeptide of 198 aa was characterized from the ovary in Nile tilapia. Multiple alignments showed that Figla was relatively conserved with the bHLH domain among fishes. Phylogenetic analysis revealed that tilapia Figla clustered into one clade with other teleostean Figla, while tetrapods Figla formed a distinct clade. The syntenic analysis showed conserved synteny of gene loci surrounding Figla gene are well conserved in vertebrates.2) Tissue distribution and ontogeny analysis were carried out by real time-PCR and IHC to analyze the expression profiles of Figla. Tissue distribution revealed that tilapia Figla gene was dominantly expressed in the XX gonads, while it was barely detectable in other tissues. Ontogenic analysis demonstrated that it was significantly up-regulated in the ovary from 30 days after hatching (dah), peaked at 3 months after hatching (mah), and decreased from 4 mah in the XX gonads. In contrast, transcript level of Figla gene remained undetectable during all stages in XY gonads. Consistently, immunohistochemistry experiments also demonstrate that Figla is expressed in the early primary oocytes at 3 and 5 mah in ovaries. On the other hand, Figla specific signals could not be detected in testes at both stages. These expression patterns show that Figla might play an important role in the differentiation and development of ovary.3) TALENs successfully and efficiently targeted (about 52.5%) Figla gene confirming by genomic PCR, restriction digestion and sequencing. Injection of TALEN lead to different bases deletions (4,8,20 etc). Furthermore, deficiency of Figla gene in XX fish led to partial sex reversal revealing by severe degeneration or complete loss of oocytes and ectopic proliferations of somatic cells in the ovary. It was shown by IHC that male specific marker genes of Leydig cells (Cyp11b2) in the testis was detected around the remanent oocytes in Figla mutated XX gonads. However, very few Cyp 19ala-positive follicular cells were detected at the same areas. At the same time, Figla knock out resulted in the continuous and sharply decrease of serum E2 levels and the increase of serum 11-KT levels. Our data indicated that mutation of Figla led to the degeneration of oocytes and differentiation of prefollicle cells into leydig cells in XX fish.4) Furthermore, over-expression of Figla gene by cloned the ORF and 3-UTR region of Figla gene into pIRES-hrGFP-1a was carried out and microinjected into XY fertilized eggs at one-cell stage. Histological observations of positive transgenic fishes revealed that Figla-transgene XY fish exhibited disruption of spermatogenesis with depletion of meiotic spermatocytes and spermatids in the testis. Dramatical decrease of Sycp3 (synaptonemal complex protein) and prm (protamine) expression indicated impaired meiotic spermatocytes and mature sperm production. Even though Sertoli cell (dmrtl) and Leydig cell (star and cypl7al) marker genes remained unaffected, hsd3bl expression and 11-KT production were raised up in Figla-transgene testis. Taken together, these data demonstrated that fish Figla might be an effective factor to repress spermatogenesis-associated genes.In summary, we isolated Figla from tilapia gonad. Multiple alignment and phylogenetic analyses revealed that Figla was relatively conserved during the vertebrate evolution. Our data indicated that mutation of Figla led to the degeneration of oocytes and transdifferentiation of follicle cells into Leydig cells in XX fish. Meanwhile, the expression of Cyp11b2 was significantly increased compared with the control XX fish. In addition, mutation of Figla in the XX fish resulted in significantly decrease of serum E2 levels and the up regulation of serum 11-kt levels compared with the control fish. Therefore, we speculated that Figla might play an essential role in the ovarian differentiation and maintenance in fish. Ectopic over-expression of Figla in XY fish led to severely disrupted spermatogenesis. In contrast to normal 3-month-old XY fish, the reduced GSI index suggested that over-expression of Figla severely impaired gonad differentiation and development. Noticeable morphological defects further supported that over-expression of Figla arrested the differentiation of spermatocytes and sperm maturation. The defective meiotic progression was further confirmed by the reduced Sycp3 and prm expression. Over-expression of female specific Figla gene in XY fish eventually arrested spermatogenesis and sperm production. Therefore, fish Figla probably might be an antagonist of genetic hierarchies of male, which in turn favors ovarian development. |
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